Stat1

Cells were lysed in RIPA buffer (Beyotime, Jiangsu, China) containing a protease inhibitor cocktail (Sigma, St. Louis, CA, USA). Total protein was assessed using a BCA Protein Assay Kit (Beyotime). Western blot analysis was performed as previously described [18 (link)]. Antibodies against the following were used in this study, PABPC1 (1:1000, Abcam, SF, USA), IFI27, HA tag, Flag tag, TSG101, CD63, CD9, PCNA, Alix, Ki67, caspase 3, CXCL10, CD34, cleaved-PARP, PARP, eIF4G, cleaved caspase 9, caspase 9, ERK, p-ERK, IFI27, STAT3, p-STAT3, STAT1, p-STAT1, NF-kB, p-NF-kB, β-actin and GAPDH (all 1:1000, Cell Signaling Technology, MA, USA), EXOSC2 and EXOSC4 (both 1:1000, Santa Cruz, MA, USA).

Human keratinocyte HaCaT cells were purchased from Elabscience (Catalog No. EP-CL-0090, Houston, TX, United States). The cells were cultured in a high-glucose-containing Dulbecco’s modified Eagle’s medium (Hyclone, Logan, UT, United States) supplemented with 1% penicillin/streptomycin (Gibco-BRL, Gaithersburg, MD, United States) and heat-inactivated 10% fetal bovine serum (Gibco-BRL) in a humidified incubator containing 5% CO₂ at 37°C. Recombinant human IFN-γ, TNF-α, Alexa 594 goat anti-rabbit antibody (cat.no. A-11037), Alexa 488 goat anti-rabbit antibody (cat.no. A-11034), and Alexa 488 goat anti-mouse antibody (cat.no. A-10680) were purchased from Thermo Fisher Scientific (Waltham, MA, United States). DRAQ5™, p-IκBα (cat.no. 2859), IκBα (cat.no. 9242), p65 (cat.no. 8242), β-actin (cat.no. 3700), p-JNK (cat.no. 9251), JNK (cat.no. 9252), p-ERK (cat.no. 9101), ERK (cat.no. 9102), p-p38 (cat.no. 9211), p38 (cat.no. 9212), p-STAT1 (Tyr) (cat.no. 9167), p-STAT1 (Ser) (cat.no. 9177), and STAT1 (cat.no. 9172) antibodies were purchased from Cell Signaling Technology (Beverly, MA, United States). PCNA (cat.no. sc-56), p50 (cat.no. sc-8414), secondary horseradish peroxidase (HRP)–conjugated anti-mouse (cat.no. sc-2357), and anti-rabbit (cat.no. sc-516102) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States).

Briefly, cells from pancreatic cell lines were washed twice with PBS and lysed in Pierce RIPA Buffer (Cat#8990, Thermo) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail 100×(Cat#78440, Thermo). Protein concentrations were quantified using Pierce BCA Protein Assay Kit (Cat#23225, Thermo). Samples were denatured at 95 °C in XT Sample Buffer 4×(Cat#1610791, Bio-Rad, Hercules, CA) and loaded onto 4–12% Criterion XT Bis-Tris Protein Gels (Cat#345-0124, Bio-Rad). Proteins were transferred onto PVDF Transfer Membrane (Cat#88518, Thermo) and probed for STING (Cat#13647S, Cell Signaling Technology, Danvers, MA), IRF3 (Cat#4302S, Cell Signaling), STAT1 (Cat#9172S, Cell Signaling), p-STAT1 (Y701) (Cat#7649S, Cell Signaling) and GAPDH (Cat#2118S, Cell Signaling). HRP-conjugated goat anti-rabbit IgG (Cat#31460, Invitrogen, Carlsbad, CA) was used as a secondary antibody. Proteins were visualized using SuperSignal West Pico PLUS Chemiluminescent Substrate (Cat#34580, Thermo).

Total proteins were extracted using SDS lysis buffer (4 %w/v SDS; 20 %v/v glycerol; 0.004 %w/v bromophenol blue; 0.125 M Tris-Cl, pH 6.8; 10 %v/v 2-mercaptoethanol) and sonication (50 kHz for 30 s; VibraCell X130PB, Sonics Materials) at 4 °C and subsequently denatured at 95 °C for 5 min. Proteins were separated on RunBlue 4-12 %w/v bis-tris polyacrylamide gels (Expedeon) and then transferred onto iBlot PVDF membranes (ThermoFisher) using the Wet/Tank Blotting Systems (Bio-Rad). Membranes were probed overnight at 4 °C in blocking solution containing the primary antibody. Primary antibodies used in this work were PARP14 (C-1) (Santa Cruz Biotechnology, sc-377150), Phospho-STAT1 (pSTAT1; Tyr701; 58D6) (Cell Signalling Technology, 9167 L), STAT1 (Cell Signalling Technology, 9172), MHC Class I H2 Kb (Abcam, ab93364), GAPDH (Proteintech, 60004-1-Ig), TAP1 (Cell Signalling Technology, 12341), TAP2 (Cell Signalling Technology, 12259; Santa Cruz Biotechnology, sc-515576), and PD-L1/B7-H1 (R&D Systems, AF1019-SP; Cell Signalling Technology, 13684). This was followed by incubation with the appropriate secondary antibody for 1.5 h at room temperature. Signals were developed using the Clarity Max Western ECL blotting substrate (Bio-Rad) and acquired on a Gel Doc XR+ Gel Documentation System (Bio-Rad). Images were analysed using Image Lab™ Software (version 3.0.1.).

Western blot assay was performed as described previously 20 (link). Briefly, total protein from cells was extracted using RIPA buffer (Beyotime, Shanghai, China) supplemented with complete EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, Basel, Switzerland). The extracted protein was separated on SDS polyacrylamide gels and transferred to the PVDF membranes. Western blotting was performed using antibodies against human COL6A1 (Abcam, Milton, Cambridge, UK), N-cadherin, E-cadherin, Vimentin, FAK, Src, p-FAK, p-Src, p-STAT1 (Tyr701), p-STAT1 (Ser727), p-STAT3 (Tyr705), STAT1, STAT3, CD133, ABC2G, SOX2, Nanog, CD63, CD9, TSG101, SOCS5, Flag, GFP, HA, Ubiqution (Ub), GAPDH, p300, c-Jun were purchased from Cell Signaling Technology (CST, USA).

Commercially available antibodies were used to detect FK2 (Enzo Life Sciences, BML-PW8810), DDK tag (Origene, TA50011), HCV NS3 (TORDJI-22, Abcam), HCV core (ThermoFisher Scientific, MA1-080), HAV capsid (Commonwealth Serum Laboratories, K24F2), Dengue capsid (Dr. Tom Hobman, University of Alberta), STAT1 (Cell Signaling Technologies, CST9175), STAT2 (Santa Cruz, sc-476), Y701-phosphoSTAT1 (Cell Signaling Technologies, 9167S), Y690-phosphoSTAT2 (Cell Signaling Technologies, 88410S), PDLIM2 (Abcam, 246868), p65 NF-κB (Santa Cruz, sc-372), Actin (EMD Millipore, MAB1501), PARP-1 (BD Pharmingen, BD556362), Lamin (Zymed, 33–2000), B-tubulin (Abcam, AB6046), Anti-NS5a (gift from Charlie Rice, 9E10). For western blotting, goat anti-mouse IR Dye 800 (Licor, 926–32210) and goat anti-rabbit IR Dye 680 (Licor, 926–32221) were used. For immunofluorescence, Alexa Fluor 546 goat anti-mouse IgG (ThermoFisher Scientific, A11030), Alexa Fluor 488 goat anti-rabbit IgG, (ThermoFisher Scientific, A11008), or Alexa Fluor 647 goat anti-mouse IgG (ThermoFisher Scientific, A21236) were used.